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R Kole

Publications and source records attributed to R Kole.

At least 37 records · Page 2Linked to original sources
PubMed1999

Antisense pharmacodynamics: critical issues in the transport and delivery of antisense oligonucleotides.

This review critically examines current understanding of the kinetics and biodistribution of antisense oligonucleotides, both at the cellular level and at the level of the intact organism. The pharmacodynamic relationships between biodistribution and the ultimate biological effects of antisense agents are considered. The problems and advantages inherent in the use of delivery systems are discussed in the light of further enhancing in vivo pharmacological actions of oligonucleotides.

Animals↗
PubMed1999

Delivery of antisense oligonucleotides and plasmid DNA with various carrier agents.

A series of cationic nucleic acid carriers was evaluated for their ability to deliver pLuc plasmid DNA or a 2'-O-methyl-oligoribonucleoside phosphorothioate, ON-705. Oligonucleotide delivery and its antisense function were assayed by a recently developed assay based on alternative splicing of modified luciferase pre-mRNA (Kang et al., 1998). This assay scores only the nuclear and sequence-specific antisense activity of the oligonucleotides. The results show that the efficiencies of delivery of plasmid DNA and oligonucleotides by the tested carriers, with the exception of Exgene and Lipofectamine, differed markedly. The efficiency of the delivery of ON-705 oligonucleotide was reduced by 70%-90% for all carriers, except Effectene, in culture media containing 8% fetal bovine serum. Interestingly, the efficiency of delivery of the ON-705-Effectene complex increased with serum concentrations of up to 30%.

Alternative Splicing↗
PubMed1999

Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations.

We have used three beta-thalassemic mutations, IVS2-654, -705 and -745, that create aberrant 5' splice sites (5' ss) and activate a common cryptic 3' ss further upstream in intron 2 of the human beta-globin gene to optimize a generally applicable exon-skipping strategy using antisense derivatives of U7 small nuclear RNA (snRNA). Introducing a modified U7 snRNA gene carrying an antisense sequence against the cryptic 3' ss into cultured cells expressing the mutant beta-globin genes, restored correct beta-globin mRNA splicing for all three mutations, but the efficiency was much weaker for IVS2-654 than for the other mutations. The length of antisense sequence influenced the efficiency with an optimum of approximately 24 nucleotides. Combining two antisense sequences directed against different target sites in intron 2, either on separate antisense RNAs or, even better, on a single U7 snRNA, significantly enhanced the efficiency of splicing correction. One double-target U7 RNA was expressed on stable transformation resulting in permanent and efficient suppression of the IVS2-654 mutation and production of beta-globin. These results suggest that forcing the aberrant exon into a looped secondary structure may strongly promote its exclusion from the mRNA and that this approach may be used generally to induce exon skipping.

Exons↗
PubMed1998

A system for shuttling 200-kb BAC/PAC clones into human cells: stable extrachromosomal persistence and long-term ectopic gene activation.

A novel shuttle vector, pBH140, has been constructed that allows stable maintenance of large genomic inserts as human artificial episomal chromosomes (HAECs) in mammalian cells. The vector, essentially a hybrid BAC-HAEC, contains an F-based replication system as in a bacterial artificial chromosome (BAC) and the Epstein-Barr virus (EBV) latent origin of replication system, oriP, for replication in human cells. A 185-kb DNA insert containing the entire human beta-globin locus, including its locus control region (LCR), was retrofitted into this vector. The resulting beta-globin BAC-HAEC clone, p148BH, was transfected into human cells and analyzed for episomal maintenance and expression of the beta-globin gene. FISH revealed an association of the vector with different human chromosomes but no integration. The beta-globin BAC-HAECs were present at an average copy number of 11-15 per nucleus in the stably transformed human cells. After 1 year of continuous in vitro cultivation, the HAECs persisted as structurally intact 200-kb episomes. While no beta-globin transcription could be detected in the parental D98/Raji cells, correctly spliced RT-PCR products were produced at significant levels in long-term cultures of the BAC-HAEC-transduced cells. The wide availability of BAC and PAC libraries, the ease in manipulating cloned DNA in bacteria, and the episomal stability of the pBH140 vector make this system ideal for studies on gene expression and other genomic functions in human cells. The potential significance of large, functionally active episomes for gene therapy is discussed.

Cell Line, Transformed↗
PubMed1998

Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development.

HeLa Tet-Off cells were transfected transiently as well as stably with a recombinant plasmid (pLuc/705) carrying the luciferase gene interrupted by a mutated human beta-globin intron 2 (IVS2-705). The mutation in the intron causes aberrant splicing of luciferase pre-mRNA, preventing translation of luciferase. However, treatment of the cells with a 2'-O-methyl-oligoribonucleotide targeted to the aberrant splice sites induces correct splicing, restoring luciferase activity. The effects are sequence-specific, depend on the concentration of the oligonucleotide, and can be modulated by the pretreatment of the cell line, Luc/705, with tetracycline. Thus, the cell line provides, among others, a novel functional assay system superior to other procedures that are based on protein down-regulation. In particular, the system would be ideal in assessing the cellular delivery efficiency of antisense oligonucleotides.

Alternative Splicing↗
PubMed1998

Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs.

In several forms of beta-thalassemia, mutations in the second intron of the beta-globin gene create aberrant 5' splice sites and activate a common cryptic 3' splice site upstream. As a result, the thalassemic beta-globin pre-mRNAs are spliced almost exclusively via the aberrant splice sites leading to a deficiency of correctly spliced beta-globin mRNA and, consequently, beta-globin. We have designed a series of vectors that express modified U7 snRNAs containing sequences antisense to either the aberrant 5' or 3' splice sites in the IVS2-705 thalassemic pre-mRNA. Transient expression of modified U7 snRNAs in a HeLa cell line stably expressing the IVS2-705 beta-globin gene restored up to 65% of correct splicing in a sequence-specific and dose-dependent manner. Cell lines that stably coexpressed IVS2-705 pre-mRNA and appropriately modified U7 snRNA exhibited up to 55% of permanent restoration of correct splicing and expression of full-length beta-globin protein. This novel approach provides a potential alternative to gene replacement therapies.

Base Sequence↗
PubMed1998

A common human beta globin splicing mutation modeled in mice.

The betaIVS-2-654 C-->T mutation accounts for approximately 20% of beta thalassemia mutations in southern China; it causes aberrant RNA splicing and leads to beta0 thalassemia. To provide an animal model for testing therapies for correcting splicing defects, we have used the "plug and socket" method of gene targeting in murine embryonic stem cells to replace the two (cis) murine adult beta globin genes with a single copy of the human betaIVS-2-654 gene. No homozygous mice survive postnatally. Heterozygous mice carrying this mutant gene produce reduced amounts of the mouse beta globin chains and no human beta globin, and have a moderate form of beta thalassemia. The heterozygotes show the same aberrant splicing as their human counterparts and provide an animal model for testing therapies to correct splicing defects at either the RNA or DNA level.

Animals↗
PubMed1997

Modification of pre-mRNA splicing by antisense oligonucleotides.

Antisene oligonucleotides have been extensively studied as agents that inhibit the expression of undesirable genes in a sequence specific manner. Results reviewed in this article show that antisene oligonucleotides can also restore the expression of genes inactivated by mutations causing genetic diseases. In this novel application, antisene oligonucleotides block aberrant splice sites created by the mutations, forcing the spliceosomes to form at correct splice sites, thus restoring the proper splicing pathway and consequently the activity of the damaged gene.

Animals↗
PubMed1996

Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides.

In one form of beta-thalassemia, a genetic blood disorder, a mutation in intron 2 of the beta-globin gene (IVS2-654) causes aberrant splicing of beta-globin pre-mRNA and, consequently, beta-globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human beta-globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human beta-globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

3T3 Cells↗
PubMed1996

Antisense 2'-O-methyloligoribonucleotides hybridized to RNA block a nuclear, ATP-dependent 3'-5' exonuclease.

RNA hybridized to 2'-O-methyloligoribonucleotides and incubated in nuclear extracts from HeLa cells is truncated, resulting in a distinct product terminated at the 5' end of the antisense oligonucleotide. The activity responsible for this effect is not RNase H but rather a novel exonuclease degrading RNA in the 3' to 5' direction. The enzymes requires ATP and Mg2+ ions. Except for dATP, no other nucleoside triphosphate or nonhydrolyzable ATP analog supports the exonucleolytic activity. In spite of the nuclear origin and activity requirements similar to those required for pre-mRNA splicing, the exonuclease operates with equal efficiency on intron-containing and intronless RNAs, excluding the possibility that it is associated with the splicing machinery.

Adenosine Triphosphate↗
PubMed1996

Effects of exon sequences on splicing of model pre-mRNA substrates in vitro.

We used several related pre-mRNA substrates consisting of two introns and three exons to study effects of exon sequences on in vitro splicing. By varying the sequence of the internal exon and measuring the frequency of its skipping we confirmed that 26-nucleotide exon element naturally existing in beta-globin gene and previously analysed in vivo, has a strong stimulatory effect on splicing. Sequence analysis of this element suggests that it belongs to a family of purine-rich splicing elements found in exons of several alternatively spliced pre-mRNAs. The 26-nucleotide element can efficiently function in enhancing inclusion of internal exons regardless of their size and sequence composition, suggesting that it plays a role of a general exon recognition element. The purine-rich element is dispensable in exons flanked by strong splice sites, which promote efficient inclusion of otherwise poorly recognized exons. A row of six cytidines inserted into the internal exon (GC2 mutation) initially considered to stimulate exon inclusion to a similar extent as the purine-rich element (Dominski & Kole, 1994, J. Biol. Chem. 269, 23590-23596), appears not to affect splice site selection in vitro, and in vivo it is likely to act by stabilizing mRNA that includes the internal exon against rapid cytoplasmic degradation.

Base Sequence↗
PubMed1995

Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5' but not the 3' splice site inhibit intron processing in Nicotiana plumbaginifolia.

We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.

Base Sequence↗
PubMed1995

Selection of novel exon recognition elements from a pool of random sequences.

A 20-nucleotide sequence close to the 3' end of the internal exon of a model two-intron, three-exon pre-mRNA (DUP184 [Z. Dominski and R. Kole, J. Biol. Chem. 269:23590-23596, 1994]) was replaced by a random 20-mer, resulting in a pool of pre-mRNAs which, like the initial DUP184 construct, were spliced in vitro by a pathway leading to predominant skipping of the internal exon. The randomized pre-mRNAs were subjected to a selection protocol, resulting in a pool enriched in pre-mRNAs that efficiently included the internal exon. Isolation and sequencing of a number of clones corresponding to the selected pre-mRNAs showed that two classes of sequences were selected from the initial pool. Most abundant among these were sequences with purine tracts similar to those in the recently identified exon-splicing enhancers while a smaller class included sequences lacking discernible purine tracts within the 20-nucleotide region. Splicing of selected pre-mRNAs showed that the purine tracts vary in their ability to promote exon inclusion and, more important, that sequences lacking purine tracts stimulate inclusion of the internal exon as efficiently as their purine-rich counterparts. The latter result indicates the existence of a novel class of exon recognition sequences or splicing enhancers.

Base Composition↗
PubMed1994

Identification of exon sequences involved in splice site selection.

The involvement of exon sequences in splice site selection was studied in vivo in HeLa cells transfected with a series of model three exon-two intron pre-mRNAs which differed only in the sequence of their internal exons. When the majority of the human globin-derived 175-nucleotide internal exon (DUP175) was replaced with a sequence from the yeast URA3 gene (DUP184), the splicing pathway changed from complete inclusion of the internal exon in DUP175 to its predominant skipping in the DUP184 construct. Skipping of the exon was reversed by increasing the strength of its flanking splicing elements indicating that exon sequences exert their effect only in the presence of relatively weak splicing signals. A series of block mutations in the internal exon of DUP184 showed that a stretch of 6 cytidine nucleotides increased the inclusion of the DUP184 internal exon about 7-fold. Mutations generating purine-rich sequences (AAG and GAAG) at the 3' end of the exon led to complete exon inclusion while stepwise insertion of sequences from the internal exon of DUP175 into the DUP184 background increased exon inclusion 5-fold. Combination of the stretch of cytidines with sequences derived from DUP175 exon resulted in complete exon inclusion indicating that diverse signals within exons may cooperate with each other in affecting splice site selection.

Base Sequence↗
PubMed1994

Hypoxia stimulates binding of a cytoplasmic protein to a pyrimidine-rich sequence in the 3'-untranslated region of rat tyrosine hydroxylase mRNA.

Reduced oxygen tension (hypoxia) induces a 3-fold increase in stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. To investigate the possibility that RNA-protein interactions are involved in mediating this increase in stability, RNA gel shift assays were performed using different fragments of labeled TH mRNA and the S-100 fraction of PC12 cytoplasmic protein extracts. We identified a sequence within the 3'-untranslated region of TH mRNA that binds cytoplasmic protein. RNase T1 mapping revealed that the protein was bound to a 28 nucleotide long sequence that is located between bases 1551-1579 of TH mRNA. Moreover, protein binding to this fragment was prevented with an antisense oligonucleotide directed against bases 1551-1579 and subsequent RNase H digestion. This fragment of the 3'-untranslated region of TH mRNA is rich in pyrimidine nucleotides, and the binding of cytoplasmic protein to this fragment was reduced by competition with other polypyrimidine sequences including poly(C) but not poly(U) polymers. The binding of the protein to TH mRNA was increased when cytoplasmic proteins were extracted from PC12 cells exposed to hypoxia (5% O2) for 24 h. Electrophoresis of the UV cross-linked RNA-protein complex on SDS-polyacrylamide gel electrophoresis revealed a complex of 74 kDa. The potential role of this protein-TH mRNA interaction in regulation of TH mRNA stability during hypoxia is discussed.

Animals↗
PubMed1994

Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.

Base Sequence↗
PubMed1993

Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides.

Antisense 2'-O-methylribooligonucleotides were targeted against specific sequence elements in mutated human beta-globin pre-mRNAs to restore correct splicing of these RNAs in vitro. The following mutations of the beta-globin gene, A-->G at nt 110 of the first intron (beta 110), T-->G at nt 705 and C-->T at nt 654 of the second intron (IVS2(705) and IVS2(654), respectively), which led to aberrant splicing of the corresponding pre-mRNAs, were previously identified as the underlying causes of beta-thalassemia. Aberrant splicing of beta 110 pre-mRNA was efficiently reversed by an oligonucleotide targeted against the branch point sequence in the first intron of the pre-mRNA but not by an oligonucleotide targeted against the aberrant 3' splice site. In both IVS2(705) and IVS2(654) pre-mRNAs, correct splicing was restored by oligonucleotides targeted against the aberrant 5' splice sites created by the mutations in the second intron or against a cryptic 3' splice site located upstream and activated in the mutated background. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective gene and not, as usual, to down-regulate the expression of an undesirable gene.

Base Sequence↗